Henry M. Miziorko

ENZYMES OF THE MEVALONATE PATHWAY OF ISOPRENOID BIOSYNTHESIS

The mevalonate pathway accounts for conversion of acetyl-CoA to isopentenyl 5-diphosphate, the versatile precursor of polyisoprenoid metabolites and natural products. The pathway functions in most eukaryotes, archaea, and some eubacteria. Only recently has much of the functional and structural basis for this metabolism been reported. The biosynthetic acetoacetyl-CoA thiolase and HMG-CoA synthase reactions rely on key amino acids that are different but are situated in active sites that are similar throughout the family of initial condensation enzymes. Both bacterial and animal HMG-CoA reductases have been extensively studied and the contrasts between these proteins and their interactions with statin inhibitors defined. The conversion of mevalonic acid to isopentenyl 5-diphosphate involves three ATP-dependent phosphorylation reactions. While bacterial enzymes responsible for these three reactions share a common protein fold, animal enzymes differ in this respect as the recently reported structure of human phosphomevalonate kinase demonstrates. There are significant contrasts between observations on metabolite inhibition of mevalonate phosphorylation in bacteria and animals. The structural basis for these contrasts has also recently been reported. Alternatives to the phosphomevalonate kinase and mevalonate diphosphate decarboxylase reactions may exist in archaea. Thus, new details regarding isopentenyl diphosphate synthesis from acetyl-CoA continue to emerge.

Identification of Catalytic Residues in Human Mevalonate Kinase

cDNA encoding human mevalonate kinase has been overexpressed and the recombinant enzyme isolated. This stable enzyme is a dimer of 42-kDa subunits and exhibits aV m = 37 units/mg,K m (ATP) = 74 μm, andK m (DL-MVA) = 24 μm. The sensitivity of enzyme to water-soluble carbodiimide modification of carboxyl groups prompted evaluation of four invariant acidic amino acids (Glu-19, Glu-193, Asp-204, and Glu-296) by site-directed mutagenesis. Elimination of Glu-19’s carboxyl group (E19A, E19Q) destabilizes the enzyme, whereas E19D is stable but exhibits only ∼2-fold changes in V m and K m values. E296Q is a stable enzyme, which exhibits kinetic parameters comparable to those measured for wild-type enzyme. E193A is a labile protein, whereas E193Q is stable, exhibiting >50-fold diminution in V m and elevatedK m values for ATP (∼20-fold) and mevalonate (∼40-fold). Such effects would be compatible with a role for Glu-193 in interacting with the cation of the MgATP substrate. D204A and D204N are stable enzymes lacking substantial mevalonate kinase activity. The active sites of these Asp-204 mutants are intact, based on their ability to bind a spin-labeled ATP analog with stoichiometries and equilibrium binding constants that are comparable to those determined for wild-type enzyme. Competitive displacement experiments demonstrate that the Asp-204 mutants can bind ATP with K d values that are comparable to estimates for wild-type enzyme. The >40,000-fold diminution in k cat for the Asp-204 mutants and the demonstration that they contain an otherwise intact active site support assignment of a crucial catalytic role to Asp-204. The assignment of Asp-204 as the catalytic base that facilitates deprotonation of the C-5 hydroxyl of mevalonic acid would be compatible with the experimental observations.

Identification of active site residues in mevalonate diphosphate decarboxylase: Implications for a family of phosphotransferases

A combination of sequence homology analyses of mevalonate diphosphate decarboxylase (MDD) proteins and structural information for MDD leads to the hypothesis that Asp 302 and Lys 18 are active site residues in MDD. These residues were mutated to replace acidic/basic side chains and the mutant proteins were isolated and characterized. Binding and competitive displacement studies using trinitrophenyl‐ATP, a fluorescent analog of substrate ATP, indicate that these mutant enzymes (D302A, D302N, K18M) retain the ability to stoichiometrically bind nucleotide triphosphates at the active site. These observations suggest the structural integrity of the mutant MDD proteins. The functional importance of mutated residues was evaluated by kinetic analysis. The 103 and 105‐fold decreases in kcat observed for the Asp 302 mutants (D302N and D302A, respectively) support assignment of a crucial catalytic role to Asp 302. A 30‐fold decrease in activity and a 16‐fold inflation of the Km for ATP is documented for the K18M mutant, indicating that Lys 18 influences the active site but is not crucial for reaction chemistry. Demonstration of the influence of conserved aspartate 302 appears to represent the first documentation of the functional importance of a residue in the MDD catalytic site and affords insight into phosphotransferase reactions catalyzed by a variety of enzymes in the galactokinase, homoserine kinase, mevalonate kinase, phosphom‐evalonate kinase (GHMP kinase) family.

Staphylococcus aureus Mevalonate Kinase: Isolation and Characterization of an Enzyme of the Isoprenoid Biosynthetic Pathway

It has been proposed that isoprenoid biosynthesis in several gram-positive cocci depends on the mevalonate pathway for conversion of acetyl coenzyme A to isopentenyl diphosphate. Mevalonate kinase catalyzes a key reaction in this pathway. In this study the enzyme from Staphylococcus aureus was expressed in Escherichia coli, isolated in a highly purified form, and characterized. The overall amino acid sequence of this enzyme was very heterologous compared with the sequences of eukaryotic mevalonate kinases. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical gel filtration chromatography suggested that the native enzyme is a monomer with a molecular mass of approximately 33 kDa. The specific activity was 12 U/mg, and the pH optimum was 7.0 to 8.5. The apparent K(m) values for R,S-mevalonate and ATP were 41 and 339 micro M, respectively. There was substantial substrate inhibition at millimolar levels of mevalonate. The sensitivity to feedback inhibition by farnesyl diphosphate and its sulfur-containing analog, farnesyl thiodiphosphate, was characterized. These compounds were competitive inhibitors with respect to ATP; the K(i) values were 46 and 45 micro M for farnesyl diphosphate and its thio analog, respectively. Parallel measurements with heterologous eukaryotic mevalonate kinases indicated that S. aureus mevalonate kinase is much less sensitive to feedback inhibition (K(i) difference, 3 orders of magnitude) than the human enzyme. In contrast, both enzymes tightly bound trinitrophenyl-ATP, a fluorescent substrate analog, suggesting that there are similarities in structural features that are important for catalytic function.

Identification and Functional Characterization of an Active-site Lysine in Mevalonate Kinase

We report the construction of an expression plasmid for rat mevalonate kinase and the overexpression of recombinant enzyme in Escherichia coli. The homogeneous enzyme had a specific activity of 30 units/mg and an observed subunit molecular mass of 42 kDa. The Michaelis constants (Km) for DL-potassium mevalonate (288 μM) and for ATP (1.24 mM) were in agreement with values reported for enzymes isolated from rat liver (Tanaka, R. D., Schafer, B. L., Lee, L. Y., Freudenberger, J. S., and Mosley, S. T. (1990) J. Biol. Chem. 265, 2391-2398). Recombinant rat mevalonate kinase was inactivated by the lysine-specific reagent, pyridoxal phosphate (PLP). ATP (5 mM) afforded protection against inactivation, suggesting reaction of PLP with an active-site lysine. Mapping, isolation, and Edman degradation of the ATP-protectable peptide from [3H]PLP-inactivated borohydride-reduced mevalonate kinase allow assignment of lysine 13, a residue invariant in known mevalonate kinase sequences, as the modification site. These results represent the first identification of an active-site residue in mevalonate kinase. The function of lysine 13 was evaluated by replacing this residue with methionine. Vm of the mutant protein is diminished by 56-fold, suggesting that lysine 13 facilitates catalysis. Kd values of wild-type and mutant proteins for ATP were determined in electron spin resonance competition experiments. The observed 56-fold diminution in affinity for the mutant enzyme supports an additional role for lysine 13 in stabilization of ATP binding.

Biochemical and Structural Basis for Feedback Inhibition of Mevalonate Kinase and Isoprenoid Metabolism.

Mevalonate kinase (MK), which catalyzes a key reaction in polyisoprenoid and sterol metabolism in many organisms, is subject to feedback regulation by farnesyl diphosphate and related compounds. The structures of human mevalonate kinase and a binary complex of the rat enzyme incubated with farnesyl thiodiphosphate (FSPP) are reported. Significant FSPP hydrolysis occurs under crystallization conditions; this results in detection of farnesyl thiophosphate (FSP) in the structure of the binary complex. Farnesyl thiodiphosphate competes with substrate ATP to produce feedback inhibition of mevalonate kinase. The binding sites for these metabolites overlap, with the phosphate of FSP nearly superimposed on ATPs beta-phosphate and FSPs polyisoprenoid chain overlapping ATPs adenosine moiety. Several hydrophobic amino acid side chains are positioned near the polyisoprenoid chain of FSP and their functional significance has been evaluated in mutagenesis experiments with human MK, which exhibits the highest reported sensitivity to feedback inhibition. Results suggest that single and double mutations at T104 and I196 produce a significant inflation of the K(i) for FSPP (approximately 40-fold for T104A/I196A). Such an effect persists when K(i) values are normalized for effects on the K(m) for ATP, suggesting that it may be possible to engineer MK proteins with altered sensitivity to feedback inhibition. Comparison of animal MK protein alignments and structures with those of a MK protein from Streptococcus pneumoniae indicates that sequence differences between N- and C-terminal domains correlate with differences in interdomain angles. Bacterial MK proteins exhibit more solvent exposure of feedback inhibitor binding sites and, consequently, weaker binding of these inhibitors.

Effect of oxygen and the lipid spin label TEMPO-laurate on fluorine-19 and proton relaxation rates of the perlluoroehemical blood substitute, FC-43 emulsion

The 19F and 1H spin-lattice relaxation (T1−1) rates of a liquid perfluorochemical, perfluorotributylamine, and its emulsified form, FC-43 emulsion, were studied using the inversion-recovery method. Both 19F and 1H relaxation rates were found to be linearly proportional to the partial pressure of oxygen, indicating that oxygen solubility in FC-43 emulsion follows Henrys law and that no specific binding between molecular oxygen and perfluorochemicals occurs. TEMPO-laurate, a water-insoluble lipid spin probe, was found to partition preferentially into perfluorotributylamine. At a concentration of 4.5 × 10−4 M, it enhanced the relaxation rate of 19F nuclei in FC-43 emulsion by 50% (in the absence of oxygen) and by 32% (in the presence of t atm oxygen) at 37°C. TEMPO-laurate also enhanced the proton relaxation rate in FC-43 emulsion by 18% (in the absence of oxygen) and by 10% (in the presence of 1 atm oxygen) at 37°C. The sensitive response of the 19F spin-lattice relaxation rate to oxygen leads to the hypothesis that this rate may be a sensitive parameter for in situ determinations of oxygen tension in blood vessels perfused with perfluorochemical blood substitutes.

Modeling of a Mutation Responsible for Human 3-Hydroxy-3-methylglutaryl-CoA Lyase Deficiency Implicates Histidine 233 as an Active Site Residue

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase is inactivated by diethyl pyrocarbonate (DEPC); activity can be fully restored by incubation with hydroxylamine. Protection against DEPC inactivation is afforded by a substrate analogue, suggesting an active site location for a DEPC target. Included in the inherited defects that map within the HMG-CoA lyase gene is a point mutation that results in an arginine substitution for histidine 233, one of only two invariant histidines. These observations prompted a functional test of the importance of His-233. The mutant lyases H233R, H233A, and H233D were overexpressed in Escherichia coli, isolated, and kinetically characterized. In H233D, DEPC targets one less histidine than was measured using wild-type lyase, supporting the assignment of wild-type lyase His-233 as one of the DEPC targets. Substitution of His-233 results in diminution of activity by ~4 orders of magnitude. Km values of the mutant lyases for both substrate HMG-CoA and activator divalent cation (Mg2+ or Mn2+) are comparable to the values measured for wild-type enzyme, indicating that these enzymes retain substantial structural integrity. This conclusion is reinforced by the observation that the affinity label, 2-butynoyl-CoA, stoichiometrically modifies the mutant lyases, indicating that they contain a full complement of active sites. In view of these data suggesting that the structures of these mutant lyases closely approximate that of the wild-type enzyme, their observed 104-fold diminution in catalytic efficiency supports assignment to His-233 of a role in the chemistry of HMG-CoA cleavage.

Identification in Haloferax volcanii of phosphomevalonate decarboxylase and isopentenyl phosphate kinase as catalysts of the terminal enzyme reactions in an archaeal alternate mevalonate pathway.

Mevalonate (MVA) metabolism provides the isoprenoids used in archaeal lipid biosynthesis. In synthesis of isopentenyl diphosphate, the classical MVA pathway involves decarboxylation of mevalonate diphosphate, while an alternate pathway has been proposed to involve decarboxylation of mevalonate monophosphate. To identify the enzymes responsible for metabolism of mevalonate 5-phosphate to isopentenyl diphosphate in Haloferax volcanii, two open reading frames (HVO_2762 and HVO_1412) were selected for expression and characterization. Characterization of these proteins indicated that one enzyme is an isopentenyl phosphate kinase that forms isopentenyl diphosphate (in a reaction analogous to that of Methanococcus jannaschii MJ0044). The second enzyme exhibits a decarboxylase activity that has never been directly attributed to this protein or any homologous protein. It catalyzes the synthesis of isopentenyl phosphate from mevalonate monophosphate, a reaction that has been proposed but never demonstrated by direct experimental proof, which is provided in this account. This enzyme, phosphomevalonate decarboxylase (PMD), exhibits strong inhibition by 6-fluoromevalonate monophosphate but negligible inhibition by 6-fluoromevalonate diphosphate (a potent inhibitor of the classical mevalonate pathway), reinforcing its selectivity for monophosphorylated ligands. Inhibition by the fluorinated analog also suggests that the PMD utilizes a reaction mechanism similar to that demonstrated for the classical MVA pathway decarboxylase. These observations represent the first experimental demonstration in H. volcanii of both the phosphomevalonate decarboxylase and isopentenyl phosphate kinase reactions that are required for an alternate mevalonate pathway in an archaeon. These results also represent, to our knowledge, the first identification and characterization of any phosphomevalonate decarboxylase.

Human mevalonate diphosphate decarboxylase: characterization, investigation of the mevalonate diphosphate binding site, and crystal structure.

Expression in Escherichia coli of his-tagged human mevalonate diphosphate decarboxylase (hMDD) has expedited enzyme isolation, characterization, functional investigation of the mevalonate diphosphate binding site, and crystal structure determination (2.4A resolution). hMDD exhibits V(max)=6.1+/-0.5 U/mg; K(m) for ATP is 0.69+/-0.07 mM and K(m) for (R,S) mevalonate diphosphate is 28.9+/-3.3 microM. Conserved polar residues predicted to be in the hMDD active site were mutated to test functional importance. R161Q exhibits a approximately 1000-fold diminution in specific activity, while binding the fluorescent substrate analog, TNP-ATP, comparably to wild-type enzyme. Diphosphoglycolyl proline (K(i)=2.3+/-0.3 uM) and 6-fluoromevalonate 5-diphosphate (K(i)=62+/-5 nM) are competitive inhibitors with respect to mevalonate diphosphate. N17A exhibits a V(max)=0.25+/-0.0 2U/mg and a 15-fold inflation in K(m) for mevalonate diphosphate. N17As K(i) values for diphosphoglycolyl proline and fluoromevalonate diphosphate are inflated (>70-fold and 40-fold, respectively) in comparison with wild-type enzyme. hMDD structure indicates the proximity (2.8A) between R161 and N17, which are located in an interior pocket of the active site cleft. The data suggest the functional importance of R161 and N17 in the binding and orientation of mevalonate diphosphate.